Method of DNA vaccination

ABSTRACT

A method of vaccinating a mammal against a disease state, comprising administrating to said mammal, within an appropriate vector, a nucleotide sequence encoding an antigenic peptide associated with the disease state; additionally administering to said mammal a compound which enhances both humoral and cellular immune responses initiated by the antigenic peptide, the compound being selected from the list contained herein, wherein the compound is preferably Tucaresol or a physiologically acceptable salt or ester thereof, where appropriate.

This is a national stage application under 35 U.S.C. 371 ofPCT/EP99/06217, filed Aug. 25, 1999, now abandoned.

FIELD OF THE INVENTION

The present invention relates to improvements in DNA vaccination and inparticular, but not exclusively, to methods of vaccinating a mammalagainst disease states, to vaccine compositions and to the use ofcertain compounds in the manufacture in medicaments.

BACKGROUND OF THE INVENTION

Traditional vaccination techniques which involve the introduction intoan animal system of an antigen which can induce an immune response inthe animal, and thereby protect the animal against infection, have beenknown for many years. Following the observation in the early 1990's thatplasmid DNA could directly transfect animal cells in vivo, significantresearch efforts have been undertaken to develop vaccination techniquesbased upon the use of DNA plasmids to induce immune responses, by directintroduction into animals of DNA which encodes for antigenic peptides.Such techniques, which are referred to as “DNA immunisation” or “DNAvaccination” have now been used to elicit protective antibody (humoral)and cell-mediated (cellular) immune responses in a wide variety ofpre-clinical models for viral, bacterial and parasitic diseases.

Research is also underway in relation the use of DNA vaccinationtechniques in treatment and protection against cancer, allergies andautoimmune diseases.

DNA vaccines usually consist of a bacterial plasmid vector into which isinserted a strong viral promoter, the gene of interest which encodes foran antigenic peptide and a polyadenylation/transcriptional terminationsequence. The gene of interest may encode a full protein or simply anantigenic peptide sequence relating to the pathogen, tumour or otheragent which is intended to be protected against. The plasmid can begrown in bacteria, such as for example E. coli and then isolated andprepared in an appropriate medium, depending upon the intended route ofadministration, before being administered to the host. Followingadministration the plasmid is taken up by cells of the host where theencoded peptide is produced. The plasmid vector will preferably be madewithout an origin of replication which is functional in eukaryoticcells, in order to prevent plasmid replication in the mammalian host andintegration within chromosomal DNA of the animal concerned.

There are a number of advantages of DNA vaccination relative totraditional vaccination techniques. Firstly, it is predicted thatbecause the proteins which are encoded by the DNA sequence aresynthesised in the host, the structure or conformation of the proteinwill be similar to the native protein associated with the disease state.It is also likely that DNA vaccination will offer protection againstdifferent strains of a virus, by generating cytotoxic T lymphocyteresponses that recognise epitopes from conserved proteins. Furthermore,because the plasmids are taken up by the host cells where antigenicprotein can be produced, a long-lasting immune response will beelicited. The technology also offers the possibility of combiningdiverse immunogens into a single preparation to facilitate simultaneousimmunisation in relation to a number of disease states.

Helpful background information in relation to DNA vaccination isprovided in (1), the disclosure of which is included herein in itsentirety by way of reference.

Despite the numerous advantages associated with DNA vaccination relativeto traditional vaccination therapies, there is nonetheless a desire todevelop adjuvant compounds which will serve to increase the immuneresponse induced by the protein which is encoded by the plasmid DNAadministered to an animal.

One reason for this is that while DNA vaccines tend to work well in micemodels, there is evidence of a somewhat weaker potency in larger speciessuch as non-human primates (2, 3), which is thought to be predictive ofthe likely potency in humans. Adjuvants may also be useful to correct aninappropriate deviation of immune response from a Th1 to Th2 responsewhich can be associated with DNA vaccination, especially whenadministered directly to the epidermis (4). Finally, it has beenrecognised that the DNA itself, through CpG motifs, may exhibit someadjuvant properties (5, 6, 7) which are prevalent in smaller animalsadministered DNA vaccines intramuscularly, but reduced in larger speciesor when small amounts of DNA are administered, such as via “gene-gun”administration.

Accordingly, it is one object of the present invention to provideadjuvant compounds which can be used in conjunction with DNA vaccinationprocedures. It is also an object to provide methods of improved DNAvaccination involving such adjuvants, as well as compositions includingthe adjuvants concerned. Other objects of the present invention willbecome apparent from the following detailed description thereof. Todate, however, meeting these objects has proven difficult, largely dueto mechanistic differences associated with DNA vaccination, as comparedto traditional vaccine techniques.

The literature reports numerous instances of humoral immune responses inanimal models, which result from DNA vaccination. Antibody responseshave been shown against human growth hormone and human α-1 anti-trypsin(8), against influenza NP (9), against HIV Envelope protein (10), bovineherpes virus glycoprotein (11) and hepatitis B surface antigen (12),amongst others, following administration of plasmid DNA encodingtherefore. Cytotoxic T-cell responses have also been demonstrated inanimal models of DNA vaccination. Generation of cytotoxic T-cells hasbeen demonstrated against NP from influenza A (13), hepatitis B surfaceantigen (HBs Ag) 65 and core antigen (14), and HIV Env (15, 16, 17) as afew examples. It is interesting to note, however, that helper T-cellresponse appears to be dependent upon the mode of plasmid DNA delivery.Intramuscular administration biases the helper T-cell response toTh1-like response, whereas administration primarily to the epidermisbiases the immune response towards a Th2-like response (4). It isfurther noted that a number of known immunopotentiating agents have beentried in combination with DNA vaccination techniques with limited, or atbest, mixed success. For example, while co-expression of GM-CSF withrabies virus glycoprotein (18) and carcinoembroytic antigen (CEA (19)),and co-expression of B7-1 and B7-2 M. tuberculosis HSP 65 (20) or CEA(19), all induced higher antibody titres than expression of the antigenalone, there is no report of an enhanced cellular immune response.Interestingly also, rabies virus glycoprotein when co-administered withDNA encoding interferon-γ, actually had an inhibitory effect on antibodyresponse (18).

With this background in mind, it is most surprising to note that thepresent inventors report adjuvant compounds which show the dual actionof not only stimulating humoral immune response, but simultaneouslystimulating the cellular immune response mechanism. The compounds whichhave been recently shown to demonstrate this remarkable adjuvantactivity in relation to DNA vaccination were disclosed in InternationalPatent Publication No. WO94/07479, in relation to theirimmunopotentitory activity. One particular compound identified by thepresent inventors as having favourable DNA vaccine adjuvant activity is4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid, also known astucaresol, which was originally described in EP 0054924. None of thecompounds now identified by the present inventors as being suited to actas adjuvants with DNA vaccines have previously been disclosed orsuggested as demonstrating the humoral and cellular immunogenic activitywhich so suits them to the role as DNA vaccine adjuvants.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention there is provided amethod of vaccinating a mammal against a disease state, comprisingadministrating to said mammal, within an appropriate vector, a DNAsequence encoding an antigenic peptide associated with the diseasestate;

additionally administering to said mammal a compound which enhances bothhumoral and cellular immune responses initiated by the antigenicpeptide, the compound being selected from:

-   4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N,N-diethyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N-isopropyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   ethyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;-   (±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid;-   methyl 3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;-   3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid;-   benzyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;-   7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;-   5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;-   5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide;-   ethyl 4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;-   5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(3-acetylamino-2-formylphenoxy)pentanoic acid;-   Aminoguanidine;-   4-(2-formyl-3-hydroxyphenoxy)butanoic acid;-   6-(2-formyl-3-hydroxyphenoxy)hexanoic acid;-   ethyl 4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;-   4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;-   2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;-   5-(2-formyl-3-hydroxy-4-methoxyphenoxy)pentanoic acid;-   3-(2-formyl-3-hydroxyphenoxy)propionitrile;-   4-Hydroxyphenylacetaldehyde;-   Phenylacetaldehyde;-   4-Methoxyphenylacetaldehyde;-   1-hydroxy-2-phenylpropane;-   3-Phenylproponionaldeyde;-   4-Nitrobenzaldehyde;-   Methyl 4-formylbenzoate;-   4-Chlorobenzaldehyde;-   4-Methyloxybenzaldehyde;-   4-Methylbenzaidehyde;-   8,10-Dioxoundecanoic acid;-   4,6-Dioxoheptanoic acid;-   Pentanedione;-   5-methoxy-1-tetralone;-   6-methoxy-1-tetralone;-   7-methoxy-1-tetralone;-   2-tetralone;-   3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;-   2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;-   2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one;-   Naringenin 4′,5,6-trihydroxyflavonone;-   4′-methoxy-2-(4-methoxyphenyl)acetophenone;-   6,7-dihydroxycoumarin;-   7-methoxy-2-tetralone;-   6,7-dimethoxy-2-tetralone;-   6-hydroxy-4-methylcoumarin;-   Homogentisic acid gamma lactone;-   6-hydroxy-1,2-naphthoquinone;-   8-methoxy-2-tetralone;    and physiologically acceptable salts thereof, where appropriate.

Preferably the nucleotide sequence is a DNA sequence.

Preferably between one and seven administrations of the compound takeplace between about 14 days prior to and about 14 days postadministration of the DNA sequence, particularly preferably betweenabout 7 days prior to and about 7 days post administration of the DNAsequence, preferably between about 1 day prior to and 1 day postadministration of the DNA sequence. Most particularly preferablyadministration of the compound is substantially simultaneous toadministration of the DNA sequence, with optional furtheradministrations of the compound in the days following administration ofthe DNA sequence.

Preferably the compound is administered at a dose of between about 0.1mg/kg and about 100 mg/kg per administration.

Preferably the mammal is a human.

Preferably the compound is 4-(2-formyl-3-hydroxyphenoxymethyl)benzoicacid.

According to another embodiment of the invention there is provided avaccine composition comprising a DNA sequence which encodes for anantigenic peptide associated with a disease state and is within anappropriate vector, and a compound which will enhance both humoral andcellular immune responses in a mammal which are initiated by theantigenic peptide, the compound being selected from:

-   4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N,N-diethyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N-isopropyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   ethyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;-   (±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid;-   methyl 3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;-   3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid;-   benzyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;-   7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;-   5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;-   5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide;-   ethyl 4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;-   5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(3-acetylamino-2-formylphenoxy)pentanoic acid;-   Aminoguanidine;-   4-(2-formyl-3-hydroxyphenoxy)butanoic acid;-   6-(2-formyl-3-hydroxyphenoxy)hexanoic acid;-   ethyl 4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;-   4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;-   2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;-   5-(2-formyl-3-hydroxy-4-methoxyphenoxy)pentanoic acid;-   3-(2-formyl-3-hydroxyphenoxy)propionitrile;-   4-Hydroxyphenylacetaldehyde;-   Phenylacetaldehyde;-   4-Methoxyphenylacetaldehyde;-   1-hydroxy-2-phenylpropane;-   3-Phenylproponionaldeyde;-   4-Nitrobenzaldehyde;-   Methyl 4-formylbenzoate;-   4-Chlorobenzaldehyde;-   4-Methyloxybenzaldehyde;-   4-Methylbenzaldehyde;-   8,10-Dioxoundecanoic acid;-   4,6-Dioxoheptanoic acid;-   Pentanedione;-   5-methoxy-1-tetralone;-   6-methoxy-1-tetralone;-   7-methoxy-1-tetralone;-   2-tetralone;-   3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;-   2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;-   2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one;-   Naringenin 4′,5,6-trihydroxyflavonone;-   4′-methoxy-2-(4-methoxyphenyl)acetophenone;-   6,7-dihydroxycoumarin;-   7-methoxy-2-tetralone;-   6,7-dimethoxy-2-tetralone;-   6-hydroxy-4-methylcoumarin;-   Homogentisic acid gamma lactone;-   6-hydroxy-1,2-naphthoquinone;-   8-methoxy-2-tetralone;    and physiologically acceptable salts thereof, where appropriate.

Preferably the compound is 4-(2-formyl-3-hydroxyphenoxymethyl) benzoicacid.

According to a further embodiment of the present invention there isprovided use of a compound in the manufacture of a medicament, whereinadministration of the compound to a mammal enhances both humoral andcellular responses initiated by an antigenic peptide associated with adisease state, the peptide being expressed as a result of administrationto said mammal of a DNA sequence encoding for the peptide;

wherein said compound is selected from:

-   4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N,N-diethyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   N-isopropyl 5-(2-formyl-3-hydroxyphenoxy)pentanamide;-   ethyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;-   (±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid;-   methyl 3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;-   3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid;-   benzyl 5-(2-formyl-3-hydroxyphenoxy)pentanoate;-   5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;-   7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;-   5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;-   5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide;-   ethyl 4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;-   5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;-   5-(3-acetylamino-2-formylphenoxy)pentanoic acid;-   Aminoguanidine;-   4-(2-formyl-3-hydroxyphenoxy)butanoic acid;-   6-(2-formyl-3-hydroxyphenoxy)hexanoic acid;-   ethyl 4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;-   4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;-   2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;-   5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;-   5-(2-formyl-3-hydroxy-4-methoxyphenoxy)pentanoic acid;-   3-(2-formyl-3-hydroxyphenoxy)propionitrile;-   4-Hydroxyphenylacetaldehyde;-   Phenylacetaldehyde;-   4-Methoxyphenylacetaldehyde;-   1-hydroxy-2-phenylpropane;-   3-Phenylproponionaldeyde;-   4-Nitrobenzaldehyde;-   Methyl 4-formylbenzoate;-   4-Chlorobenzaldehyde;-   4-Methyloxybenzaldehyde;-   4-Methylbenzaldehyde;-   8,10-Dioxoundecanoic acid;-   4,6-Dioxoheptanoic acid;-   Pentanedione;-   5-methoxy-1-tetralone;-   6-methoxy-1-tetralone;-   7-methoxy-1-tetralone;-   2-tetralone;-   3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;-   2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;-   2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one;-   Naringenin 4′,5,6-trihydroxyflavonone;-   4′-methoxy-2-(4-methoxyphenyl)acetophenone;-   6,7-dihydroxycoumarin;-   7-methoxy-2-tetralone;-   6,7-dimethoxy-2-tetralone;-   6-hydroxy-4-methylcoumarin;-   Homogentisic acid gamma lactone;-   6-hydroxy-1,2-naphthoquinone;-   8-methoxy-2-tetralone;    and physiologically acceptable salts thereof, where appropriate.

Preferably the compound is 4-(2-formyl-3-hydroxyphenoxymethyl)benzoicacid and it is preferably administered at a dose of between about 0.1mg/kg and about 100 mg/kg per administration.

According to a further emobodiment of the invention there is provided acombination of components for separate, sequential or concomitantadministration in a method as outlined above, comprising the nucleotidesequence encoding an antigenic peptide and the compound which enhancesboth cellular and humoral immune responses initiated by the antigenicpeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1.

Proliferation of lymph node T cells in response to Ova peptide in vitro(in presence of 0.5% mouse serum). Results show T cell response forovalbumin in PBS (no adjuvant) and PBS alone, Complete Freunds Adjuvant(CFA) alone and combined with Ova peptide, Bacterial Lipopolysaccharide(LPS) alone and combined with Ova peptide and Bacillus Calmette Geurin(HK-BCG) alone and combined with Ova peptide.

FIG. 2.

Proliferation of lymph node T cells in response to pDNA or DNA encodingovalbumin (PVAC1.Ova) in isolation and in combination with adjuvantsLPS, CFA, GM-CSF and Heat-Killed Listeria monocytogenes (HKLM).

FIG. 3.

Effects of tucaresol on the specific antibody response to mycobacterialhsp65 after immunisation with pDNA expressing Mhsp65. Mice wereimmunised with 20 μg pDNA at two occasions, three weeks apart. Two weekslater they were bled and specific IgG (a), IgG2a (b) and IgG1 (c)anti-recombinant M.hsp65 protein were detected by ELISA. p*≧0.1 Nosignificant difference in the specific antibody response. p**≦0.05 forcomparison between p3 or p3,T and p3M.65 and between p3M.65 and p3M.65 Gor p3M.65, T in (a), between p3 or p3,T and p3M.65 or p3M.65 G in (b),and between p3 and or p3,T and p3M.65Gub (c). p***≦0.003 for comparisonbetween p3 or p3, T and p3M.65 G or p3M.65,T in (a), for comparisonbetween p3 and or p3,T and p3M.65, t and between p3M.65 and p3M.65, t in(b), and between p3 and or p3, T and p3M.65, T in (c). p and h aresignificant as compared to p3 and p3M.65 respectively.

FIG. 4.

Effect of tucaresol on the proliferative T cell response. Splenocytesfrom pDNA immunised mice were cultured in the presence of either S6c-E4alone or infected with vaccinia expressing EBNA-4 9S6C-VE4) or S6C-gptcontrol tumor cells alone or infected with vaccinia TK- control vacciniaconstruct 9S6C-gptV). Stimulation index was calculated as described inthe methods section.

FIG. 5.

IFNγ response in pDNA immunised mice and the effect of tucaresol on it.Groups of mice were immunised with p3, E4 or E4, T and splenocytes werestimulated in vitro with tumor S6C-E$, S6C-gpt or nothing for 72h. IFNγtires were determined by specific ELISA.

FIG. 6.

Cytotoxic T-cell response is markedly enhanced in tucaresol treatedmice. Groups of HLA-A2/kb transgenic mice were immunised twice withp3M.65, p3M.65γ or p3M.65, T. Splenocytes were cultured with HLA-A2biding peptide P$ for 5–6 days and were then used as effectors inconventional ⁵¹Cr release assay. Using Jk-A2 kb as targets pulsed withP4, with an irrelevant peptide (Inf) or with nothing as described in themethods section.

FIG. 7.

Tumour outgrowth inhibition is markedly enhanced in tucaresol treatedmice. Groups of ACA mice were immunised three times with p3, E4 or E4,T,and were subsequently challenged with 104 S6C tumour cells. Mice weresacrificed when tumour diameter reached 20 mm.

FIG. 8.

IFNγ production is enhanced in tucaresol treated mice following gene gunimmunisation.

C57BL/6 mice or ACA mice were immunised by gene gun with pVAC1.PR (FIG.8 a) or EBNA-4 (FIG. 8 b) respectively either alone or in combinationwith tucaresol, or the respective negative plasmids. Splenocytes werecultured with a nucleoprotein peptide, or tumour cells expressing theEBNA-4 antigen, or antigen negative tumour cells. IFNγ titres weredetermined by specific ELISPOT assay (FIG. 8 b) or by specific ELISAassay (FIG. 8 a).

FIG. 9.

Tucaresol Enhances the lytic CTL response induced by gene gun DNAimmunisation.

C57B1/6 mice were immunised with plasmid DNA encoding the influenzavirus nucleoprotein with or without tucaresol given subcutaneously (FIG.9 a) or orally (FIG. 9 b). Splenocytes were restimulated in-vitro withA/PR8/34 virus. Standard europium release techniques were used todetermine specific lysis of MHC matched target cells pulsed with H-2D^(b)-restricted NP peptide.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the appended claims, unless thecontext requires otherwise, the words “comprise” and “include” orvariations such as “comprising”, “comprises”, “including”, “includes”,etc., are to be construed inclusively, that is, use of these words willimply the possible inclusion of integers or elements not specificallyrecited.

As described above, the present invention relates to vaccinationmethods, and in particular to improvements of methods of vaccinationinvolving the introduction into a mammal of DNA which encodes for anantigenic protein or peptide, such that the protein or peptide will beexpressed within the mammalian body to thereby induce an immune responsewithin the mammal, against the antigenic protein or peptide. Suchtechniques are well known and are fully described in (1) as referred toabove.

It is possible for the vaccination methods according to the presentapplication to be adapted for protection of mammals against a variety ofdisease states such as, for example, viral, bacterial or parasiticinfections, cancer, allergies and autoimmune disorders. Some specificexamples of disorders or disease states which can be protected againstor treated by using the methods or compositions according to the presentinvention, are as follows:

Viral Infections

Hepatitis viruses A, B, C, D & E, HIV, herpes viruses 1, 2, 6 & 7,-cytomegalovirus, varicella zoster, papilloma virus, Epstein Barr virus,influenza viruses, para-influenza viruses, adenoviruses, coxsakieviruses, picorna viruses, rotaviruses, respiratory syncytial viruses,pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus,measles virus.

Bacterial Infections

Mycobacteria causing TB and leprosy, pneumocci, aerobic gram negativebacilli, mycoplasma, staphyloccocal infections, streptococcalinfections, salmonellae, chlamydiae.

Parasitic

Malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis,filariasis,

Cancer

Breast cancer, colon cancer, rectal cancer, cancer of the head and neck,renal cancer, malignant melanoma, laryngeal cancer, ovarian cancer,cervical cancer, prostate cancer.

Allergies

Rhinitis due to House dust mite, pollen and other environmentalallergens

Autoimmune disease

Systemic lupus erythematosis

It is to be recognised that these specific disease states have beenreferred to by way of example only, and are not intended to be limitingupon the scope of the present invention.

The DNA sequences referred to in this application, which are to beexpressed within a mammalian system, in order to induce an antigenicresponse, may encode for an entire protein, or merely a shorter peptidesequence which is capable of initiating an antigenic response.Throughout this specification and the appended claims, the phrase“antigenic peptide” is intended to encompass all peptide or proteinsequences which are capable of inducing an immune response within theanimal concerned. Most preferably, however, the DNA sequence will encodefor a full protein which is associated with the disease state, as theexpression of full proteins within the animal system are more likely tomimic natural antigen presentation, and thereby evoke a full immuneresponse. Some non-limiting examples of known antigenic peptides inrelation to specific disease states include the following:

HBV—PreS1 PreS2 and Surface env proteins, core and pol

HIV—gp120 gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef

Papilloma—E1, E2, E3, E4, E5, E6, E7, E8, L1, L2

HSV—gL, gH, gM, gB, gC, gK, gE, gD, ICP47, ICP36, ICP4

Influenza—haemaggluttin, nucleoprotein

TB—Mycobacterial super oxide dismutase, 85A, 85B, MPT44, MPT59, MPT45,HSP10, HSP65, HSP70, HSP90, PPD 19 kDa Ag, PPD 38 kDa Ag.

In order to obtain expression of the antigenic peptide within mammaliancells, it is necessary for the DNA sequence encoding the antigenicpeptide to be presented in an appropriate vector system. For example,the vector selected may comprise a bacterial plasmid and a strong viralpromoter and polyadenylation/transcriptional termination sequencearranged in the correct order to obtain expression of the antigenicpeptides. The construction of vectors which include these components andoptionally other components such as enhancers, restriction enzyme sitesand selection genes, such as antibiotic resistance genes, is well knownto persons skilled in the art and is explained in detail in Maniatis etal (21).

As it is important to prevent the plasmids replicating within themammalian host and integrating within the chromosomal DNA of the animal,the plasmid will preferably be produced without an original ofreplication that is functional in eukaryotic cells.

The methods and compositions according to the present invention can beused in relation to prophylactic or treatment procedures of all mammalsincluding, for example, domestic animals, laboratory animals, farmanimals, captive wild animals and most preferably, humans.

The compounds recited above which have been identified by the presentinventors as exhibiting the favourable activity of enhancing bothhumoral and cellular immunogenic activity initiated by DNA vaccineadministration are known compounds, previously reported in WO94/07479 ashaving immunopotentiatory properties. In particular, the preferredcompound 4-(2-formol-3-hydroxyphenoxymethyl)benzoic acid, which is alsoknown as tucaresol, was originally described in EP 0054924 and has beenreported as having immunopotentiatory activity and as being useful fortreatment of various disorders including HIV, HBV, HCV, tumours andsickle cell anaemia. It is thought that the reported activity oftucaresol can be explained by its ability to form Schiff bases and thatit can thereby substitute for physiological donors or carbonyl groupsand provide a co-stimulatory signal to CD4 Th-cells. It was previouslyreported that tucaresol enhanced CD4 Th-cell response, selectivelyfavouring a Th 1-type profile over Th 2 (22), whereas the presentinventors have demonstrated that it is capable of enhancing both Th 1and Th 2 isotypes of antibody in a murine model.

By referring to enhancement of both humoral and cellular immuneresponses initiated by the antigenic peptide, and caused by thecompounds of the present invention, it is intended to convey that bothserum antibody levels and cytotoxic T lymphocyte (CTL) levelsrespectively will be raised as a result of administration of thecompounds, compared to levels associated with administration of DNAsequence encoding for the antigenic peptide alone. Such levels can bequantified by methods well known in the art, as will be furtherexplained in the appended examples.

The vectors which comprise the DNA sequences encoding antigenic peptidescan be administered in a variety of manners. It is possible for thevectors to be administered in a naked form (that is as naked DNA not inassociation with liposomal formulations, with viral vectors ortransfection faciltating proteins) suspended in an appropriate medium,for example a buffered saline solution such as PBS and then injectedintramuscularly, subcutaneously, intraperitonally or intravenously,although some earlier data suggests that intramuscular or subcutaneousinjection is preferable (23), (the disclosure of which is includedherein in its entirety by way of reference). It is additionally possiblefor the vectors to be encapsulated by, for example, liposomes or withinpolylactide co-glycolide (PLG) particles (25) for administration via theoral, nasal or pulmonary routes. It is also possible, according to apreferred embodiment of the invention, for intradermal administration ofthe vector, preferably via use of gene-gun (particularly particlebombardment) administration techniques. Such techniques may involvelyophilisation of a suspension comprising the vector and subsequentcoating of the vector on to gold beads which are then administered underhigh pressure into the epidermis, such as, for example, as described in(26). The amount of DNA delivered will vary significantly, dependingupon the species and weight of mammal being immunised, the nature of thedisease state being treated/protected against, the vaccination protocoladopted (i.e. single administration versus repeated doses), the route ofadministration and the potency and dose of the adjuvant compound chosen.Based upon these variables, a medical or veterinary practitioner willreadily be able to determine the appropriate dosage level.

It is possible for the DNA vector, including the DNA sequence encodingthe antigenic peptide, to be administered on a once off basis or to beadministered repeatedly, for example, between 1 and 7 times, preferablybetween 1 and 4 times, at intervals between about 1 day and about 18months. Once again, however, this treatment regime will be significantlyvaried depending upon the size and species of animal concerned, thedisease which is being treated/protected against, the amount of DNAadministered, the route of administration, the potency and dose ofadjuvant compound selected and other factors which would be apparent toa skilled veterinary or medical practitioner.

The adjuvant compound specified herein can similarly be administered viaa variety of different administration routes, such as for example, viathe oral, nasal, pulmonary, intramuscular, subcutaneous, intradermal ortopical routes. This administration may take place between about 14 daysprior to and about 14 days post administration of the DNA sequence,preferably between about 7 days prior to and about 7 days postadministration of the DNA sequence, more preferably between about 24hours prior to and 24 hours post administration of the DNA sequence, andparticularly preferably, substantially simultaneous with administrationof the DNA sequence. By “substantially simultaneous” what is meant isthat administration of the compound is preferably at the same time asadministration of the DNA sequence, or if not, at least within a fewhours either side of DNA sequence administration. In the most preferredtreatment protocol, the compound will be administered substantiallysimultaneously to administration of the DNA sequence, and then again onapproximately a daily basis for up to 14 days post DNA sequenceadministration, preferably daily for the 3 days following initialadministration. Obviously, this protocol can be varied as necessary, inaccordance with the type of variables referred to above. Once again,depending upon such variables, the dose of administration will alsovary, but may, for example, range between about 0.1 mg per kg to about100 mg per kg, where “per kg” refers to the body weight of the mammalconcerned. This administration of the adjuvant compound would preferablybe repeated with each subsequent or booster administration of the DNAsequence. Most preferably, the administration dose will be between about0.1 mg per kg to about 10 mg per kg, preferably between about 1 mg perkg and about 5 mg per kg.

While it is possible for the adjuvant compounds to be administered inthe raw chemical state, it is preferable for administration in the formof a pharmaceutical composition. That is, the compounds will preferablybe combined with one or more pharmaceutically or veterinarily acceptablecarriers, and optionally other therapeutic ingredients. The carrier(s)must be “acceptable” in the sense of being compatible with otheringredients within the formulation, and not deleterious to the recipientthereof. The nature of the formulations will naturally vary according tothe intended administration route, and may be prepared by methods wellknown in the pharmaceutical art. All methods include the step ofbringing into association a compound of the invention (the adjuvantcompound) with an appropriate carrier or carriers. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the compound with liquid carriers or finely divided solidcarriers, or both, and then, if necessary, shaping the product into thedesired formulation. Formulations of the present invention suitable fororal administration may be presented as discrete units such as capsules,cachets or tablets each containing a pre-determined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent.

The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active ingredient.

Formulations for injection via, for example, the intramuscular,intraperitonile, or subcutaneous administration routes include aqueousand non-aqueous sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described. Formulations suitable forpulmonary administration via the buccal or nasal cavity are presentedsuch that particles containing the active ingredient, desirably having adiameter in the range of 0.5 to 7 microns, are delivered into thebronchial tree of the recipient. Possibilities for such formulations arethat they are in the form of finely comminuted powders which mayconveniently be presented either in a piercable capsule, suitably of,for example, gelatine, for use in an inhalation device, oralternatively, as a self-propelling formulation comprising activeingredient, a suitable liquid propellant and optionally, otheringredients such as surfactant and/or a solid diluent. Self-propellingformulations may also be employed wherein the active ingredient isdispensed in the form of droplets of a solution or suspension. Suchself-propelling formulations are analogous to those known in the art andmay be prepared by established procedures. They are suitably providedwith either a manually-operable or automatically functioning valvehaving the desired spray characteristics; advantageously the valve is ofa metered type delivering a fixed volume, for example, 50 to 100 μL,upon each operation thereof.

In a further possibility, the active ingredient may be in the form of asolution for use in an atomiser or nebuliser whereby an acceleratedairstream or ultrasonic agitation is employed to produce a find dropletmist for inhalation.

Formulations suitable for intranasal administration generally includepresentations similar to those described above for pulmonaryadministration, although it is preferred for such formulations to have aparticle diameter in the range of about 10 to about 200 microns, toenable retention within the nasal cavity. This may be achieved by, asappropriate, use of a powder of a suitable particle size, or choice ofan appropriate valve. Other suitable formulations include coarse powdershaving a particle diameter in the range of about 20 to about 500microns, for administration by rapid inhalation through the nasalpassage from a container held close up to the nose, and nasal dropscomprising about 0.2 to 5% w/w of the active ingredient in aqueous oroily solutions.

Examples of appropriate formulations which comprise the adjuvantcompounds according to the present invention are provided withinWO94/07479, the disclosure of which is included herein by reference, inits entirety.

In a preferred embodiment of the invention, it is possible for thevector which comprises the DNA sequence encoding the antigenic peptideto be administered within the same formulation as the adjuvant compound.In a particularly preferred embodiment the adjuvant compound is preparedin a form suitable for gene-gun administration, and is administrated viathat route substantially simultaneous to administration of the DNAsequence. For preparation of formulations suitable for use in thismanner, it may be necessary for the adjuvant compound to be lyophilisedand adhered onto, for example, gold beads which are suited for gene-gunadministration.

Even if not formulated together, it may be appropriate for the adjuvantcompounds to be administered at or about the same administration site asthe DNA sequence.

Other details of pharmaceutical preparations can be found in (24), thedisclosure of which is included herein in its entirety, by way ofreference.

The present invention will now be described further, with reference tothe following non-limiting examples:

EXAMPLE 1

Activity in Conventional Adjuvants in DNA Vaccination Compared toPeptide Immunisation

Lymphoid cells from TCR-transgenic mice, expressing a receptor specificfor an ovalbumin antigen were transferred into normal syngeneic mice.These mice were then immunised subcutaneously with ovalbumin peptidewith or without adjuvants. Three days later, regional lymph node cellswere removed and their proliferative response to ovalbumin peptide wasmeasured by means of incorporation of tritiated thymidine into DNA. Thisprovides a measure of the degree of specific T-cell priming thatoccurred in vivo in response to immunisation. Immunisation withovalbumin peptide alone (◯) resulted in a significant but low level ofT-cell priming compared with mock immunisation (●). Bacteriallipopolysaccharide (LPS) (

), complete Freunds adjuvant (CFA) (□), and Bacillus Calmette Geurin(HK-BCG) (⊕), all provided significant enhancement of this response. Inthe absence of an immunising antigen, administration of bacteriallipopolysaccharide (LPS) (▴), complete Freunds adjuvant (CFA) (▪), andBacillus Calmette Geurin (HK-BCG) (▾), had minimal effects on subsequentproliferation in response to ova peptide.

Lymphoid cells from TCR-transgenic mice, expressing a receptor specificfor an ovalbumin antigen were transferred into normal syngeneic mice.These mice were then immunised subcutaneously (by means of gene gun)with plasmid DNA (pVAC1) or with plasmid DNA construct encodingovalbumin (pVAC1.Ova) with or without adjuvants given subcutaneously.Three days later, regional lymph node cells were removed and theirproliferative response to ovalbumin peptide was measured by means ofincorporation of tritiated thymidine into DNA. This provides a measureof the degree of specific T-cell priming that occurred in vivo inresponse to immunisation. A control group were immunised with ovalbuminpeptide in complete Freunds adjuvant which produced substantial T-cellpriming (♦). DNA immunisation with pVAC1.Ova (

) produced significant T-cell priming compared with immunisation withthe empty vector (◯). However, in contrast to the conventional peptideimmunisation, none of the adjuvants were able to enhance this responseto DNA immunisation. LPS (▴), heat-killed Listeria monocytogenes (HKLM)(▾), GM-CSF (⋄), and complete Freunds adjuvant (CFA) (⊕), all failed toenhance the response to DNA vaccination. [An anomalous high response wasseen in with the empty vector control using HKLM as adjuvant].

EXAMPLE 2

Tucaresol Enhances the Production of Antigen Specific Antibodies

The effect of tucaresol on immunisation with a plasmid DNA coding forthe mycobacterial heat shock protein 65 (M.hsp65) antigen was analysed,and compared the effect of tucaresol to that of plasmids expressing thecytokines GM-CSF and IFNγ.

Groups of mice were immunised intramuscularly (i.m.) with 20 μg of aplasmid (p3). Significant amounts of antibodies to M>hsp65 could bedetected in sera from p3M.65 immunised mice, but not in the p3 immunisedones (FIG. 3 a). The antibody titres were markedly increased when 1 mgof tucaresol was administered subcutaneously simultaneously with theM.hsp plasmid 9p3M.65, T). In contrast, no increase in the specificantibody response was detected in a group of mice immunised with thecontrol plasmid and tucaresol (p3, T), excluding the possibility that ageneral increase in non-specific cross-reactive antibodies due to thehigh degree of immuno-potentiation associated with tucaresoladministration accounted for the observed effect. (FIG. 3 a).

We also compared in the same experiment the effect of tucaresol withthat of injecting plasmids expressing the cytokines GM-CSF and IFNγ. Thep3M.65 plasmid was administered alone or in equimolar combination witheither a GM-CSF expressing plasmid (p3M.65, G), an IFNγ expressingplasmid (p3M.65, γ), or a mixture of GM-CSF and IFNγ expressing plasmids(p3M.65, Gγ). The anti-M.hsp65 titres were markedly increased when theGM-CSF plasmid was included in the immunisation as compared with top3M65. In contrast to the potentiating effect of the GM-CSF plasmid onthe M.hsp65 specific antibody response, there was insignificant antibodyresponse when the IFNγ expressing plasmid was included in line withpreviously published data. Combining both cytokine plasmids seemed toantagonise the enhancing effect of the GM-CSF plasmid, reducing theresponse to levels essentially similar to that observed when immunisingwith p3M.65 only (data not shown). Collectively, these resultsdemonstrate that tucaresol has a potent capacity to enhance the antibodyresponse induced by genetic immunisation with the M.hsp65 antigen,comparable to the effect of the GM-CSF plasmid.

We have analysed the isotype of the anti-M.hsp65 antibodies. p3M.65immunised mice produced significant amounts of IgG2a anti-M.hsp65antibodies. This titre was significantly (P=0.003) increased in micereceiving tucaresol (FIG. 3 b). This clear enhancement of a Th1associated antibody response was unique as immunisation with p3M.65,G,p3M.65γ or p3M.65 Gγ could not exert such an effect. The inclusion ofthe IFNγ expressing plasmid in the immunisation did not enhance theIgG2a anti-M.hsp65 antibody response.

A Th2 associated anti-M.hsp65 IgG1 antibody response could not bedetected in a significant amount of mice receiving p3M.65,G, p3M.65γ orp3M.65 Gγ. This response was induced in a significant amount (P=0.003)however, in mice receiving p3M.65, T and to a lesser extent (P=0.027) inpM.65G immunised mice as compared to p3 immunised mice (FIG. 3 c).

Taken together, our data prove that a significant increase in thespecific antibody response as a result of genetic immunisation could beachieved by the administration of tucaresol. Although thisadministration strongly enhanced the Th1 associated response, it didalso induce a Th2 associated antibody response. This is the first timetucaresol is reported to enhance the production of a specific antibodyresponse.

EXAMPLE 3

Tucaresol Enhances the Specific T-Cell Proliferative Response.

We next analysed the effect of tucaresol on the proliferative T-cellresponse induced by vaccination with pDNA expressing the Epstein BarrVirus (EBV) nuclear antigen number 4 (EBNA-4). Groups of mice wereimmunised i.m. with control plasmid p3 or with the EBNA-4 expressingplasmid (E4) or with E4 plus treatment with tucaresol (E4, T). A minimalproliferative response was detected in the splenocyte cultures from E4immunised mice when stimulated with the syngeneic EBNA-4 transfectedcarcinoma line (S6C-E4). Interestingly, a much stronger proliferativeresponse to S6C-E4 was obtained with splenocytes from mice immunisedi.m. with the E4 and treated s.c. with tucaresol (FIG. 4). EBNA-4vaccinia infected stimulators (S6C-VE4) also induced a higherproliferative response than S6C-E4 in the splenocytes from both E4immunised and the E4, T immunised mice as previously reported.Proliferation was calculated as simulation index (SI) using the formula:SI=splenocyte proliferation towards S6C-EBNA-4 transfectant (EBNA-4vaccinia infected)/splenocyte proliferation towards S6C-gpt(TK-vacccinia infected) control transfectant. Again, this response wasantigen focused as there was no detectable proliferation in thesplenocytes from control immunised mice (p3) towards S6C-E4 or EBNA-4vaccinia infected cells above that toward S6C-gpt or control TK-vacciniainfected S6c-gptV respectively (FIG. 4).

EXAMPLE 4

Tucaresol Significantly Enhances the Production of Th-1 Cytokines.

pDNA immunisation is associated with a predominant Th-1 response ascharacterised by production of Th1 cytokines including IFNγ. To assessthe capacity of tucaresol to enhance this Th-1 response, mice wereimmunised i.m. with p3, E4 or E4 with simultaneous administration oftucaresol s.c. Little, if any IFNγ was produced by splenocytes from miceimmunised with E4 only in response to specific stimulation with S6C-E4as compared to production from control splenocytes from p3 immunisedmice or the production in response to S6C-gpt. Interestingly,splenocytes from form mice immunised with e4 and treated with tucaresolproduced the highest amounts of IFNγ in response to specific in vitrostimulation with S6C-E4 but did not in response to control stimulationwith S6C-gpt. We were unable to detect the Th2 cytokine IL-4 productionin response to specific stimulation in splenocytes cultures from anygroup (data not shown). We therefore conclude that tucaresoladministration together with pDNA vaccination is a highly efficient wayof promoting a specific Th1 dominated cytokine response.

EXAMPLE 5

Augmentation of the Specific CTL Response by Tucaresol

To investigate the effect of tucaresol on the specific cytotoxic T-cell(CTL) response induced by pDNA vaccination, we have immunised HLA A2transgenic mice twice with either p3 M.65, p3M.65γ or p3M.65, T. Twoweeks after the last immunisation splenocytes form the immunised micewere stimulated once with the HLA-A2 restricted peptide epitope derivedfrom the mycobacterial hsp65 molecule, and the cultures tested forspecific CTL activity against the HLA-A2/kb Jurkat (Jk-A2/kbds) cellline unpulsed or pulsed with the cognate peptide or with a control HLAA2 restricted influenza peptide. Splenocytes from mice immunised withp3M.65 and tucaresol (p3M.65,T) developed high CTL activity againsttarget cells pulsed with the cognate M.hsp65 epitope while their lyticactivity against Jk-A2/kb cells unpulsed or pulsed with the controlinfluenza peptide was much lower (FIG. 6). In contrast, splenocytes frommice immunised with the p3M.65 without any co-stimulatory agent werealmost totally inactive. The inclusion of the IFNγ plasmid together withthe p3M.65 vaccine (p3M.65γ) enhanced the CTL activity of thesplenocytes as compared to the activity of splenocytes derived from themice immunised with p3M.65 only, but this cytotoxicity was only 30–50%of that observed with splenocytes form mice treated with tucaresol. Wetherefore conclude that tucaresol is a very efficient agent to enhancethe development of specific CTL when given together with pDNAvaccination.

EXAMPLE 6

Effect of Tucaresol on Tumour Outgrowth Inhibition In Vivo FollowingImmunisation with a Plasmid Expressing the Epstein Barr Virus NuclearAntigen 4 (EBNA-4)

Epstein Barr virus has been implicated in some cancers. We analysed theeffect of tucaresol on tumour outgrowth inhibition in vivo as a resultof pDNA vaccination with a plasmid expressing EBNA-4 (FIG. 7). Mice wereimmunised by intramuscular injection with 40 μg of either control mockplasmid pCDNA3 (P3), plasmid expressing EBNA-4 (E4) or plasmidexpressing EBNA-4 followed 1, 2, 3 and 4 days later with administrationof 200 μg of tucaresol s.c.—i.e. 800 μg total tucaresol per mouse (E4T).This immunisation schedule was repeated 1 month and 2 months after theinitial immunisation. Two weeks after the last immunisation mice werechallenged with 104 S6C-E4 tumour cells s.c. Mice were sacrificed whenthe tumour reached a 20 mm diameter. FIG. 7 shows that tucaresolsignificantly enhances the ability of EBNA-4 to inhibit tumouroutgrowth.

EXAMPLE 7

Effect of Tucaresol on CTL Cytokine Response Induced by Gene Gun DNAImmunisation in Mice.

The production of interferon gamma (IFNγ) by cytotoxic T lymphocytes(CTL) is a key measure of cell-mediated immune responses important inthe elimination of viral infection.

Using Influenza NP Antigen (FIG. 8 a)

C57BL/6 mice were immunised by gene gun with DNA plasmids encodingA/PR8/34 influenza virus nucleoprotein (pVAC1.PR) at two dose levels (10& 100 ng) or empty vector. Spleens were collected 14 days postimmunisation and splenocytes re-stimulated in vitro with an NP peptide(10 μM), recognised only by CD8 cytotoxic T-cells, together withrecombinant human IL-2 (50 ng/ml). IFNγ positive cells per 10e6splenocytes were detected by ELISPOT assay which measures the number ofindividual cells producing cytokine (mean±S.E.M.; n=3 mice). Tucaresolwas administered to mice at the time of immunisation eithersubcutaneously (s.c.) (2×1 mg) at the site of intra-epidermal DNAvaccination, or by oral gavage (15 mg/kg) daily for 5 days beginning onthe day of immunisation. Subcutaneous tucaresol produces a small butsignificant increase in the CTL cytokine response to immunisation whileoral tucaresol produces a doubling of the response.

Using Epstein Barr Virus Nuclear Antigen 4 (EBNA-4) (FIG. 8 b)

Mice were immunised by gene gun with DNA plasmids encoding EBNA-4 (E4)or empty vector (P3). 2 ug of DNA was administered per shot, giving twonon overlapping shots per mouse. Tucaresol was administered to the mice(E4T) in 200 ug amounts subcutaneously on days 1, 2, 3 and 4. Mice wereboosted after two months with the same immunisation and treatmentschedule. Two weeks later splenocytes were stimulated in vitro withtumour cells expressing the EBNA-4 antigen (S6C-E4) or with antigennegative tumour cells (S6C-gpt) for 72 h. Supernatants were thencollected and IFN-y titres were determined by specific ELISA. Tucaresoldramatically increased the production of IFNγ.

EXAMPLE 8

Effect of Tucaresol on Lytic CTL Response Induced by Gene Gun DNAImmunisation

Lysis of target cells by CD8 CTL is a principal mechanism in theelimination of viral infections by the immune system. Lytic CTL can bemeasured using target cells carrying viral peptide and labelled witheuropium.

Subcutaneous Administration of Tucaresol (FIG. 9 a)

C57BI/6 mice were immunised with plasmid DNA (10 ng) encoding theinfluenza virus nucleoprotein (NP) with and without tucaresol givensub-cutaneously (sc). Mice were killed 14 days post-immunisation andrestimulated in-vitro (5 days) with irradiated splenocytes pulsed withvirus (A/PR8/34). Standard europium release techniques were used todetermine specific lysis of MHC-matched target cells (EL4 cells) pulsedwith H-2 D^(b)-restricted NP peptide. Non-specific lysis was less than15% for all controls. Tucaresol was given subcutaneously (1 mg) at thesite of intrepidermal gene-gun immunisation.

Oral Administration of Tucaresol (FIG. 9 b)

C57B1/6 mice were immunised with plasmid DNA (10 ng) encoding theinfluenza virus nucleoprotein (NP) with and without tucaresol givenorally. Mice were killed 14 days post-immunisation and restimulatedin-vitro (5 days) with irradiated splenocytes pulsed with virus(A/PR8/34). Standard europium release techniques were used to determinespecific lysis of EL4 cells pulsed with H-2D^(b)-restricted NP peptide.Non-specific lysis was less than 15% for all controls. Tucaresol (15mg/kg) was given by oral gavage once daily for 5 days beginning on theday of immunisation.

Discussion of Experimental Results

We introduce herein a simple and very effective approach to enhance pDNAimmunisation, based on providing a co-stimulatory signal to T-cells viaSchiff base formation. Several of our observations demonstrate that thismethod is able to circumvent the problem of limitation in efficacy whichcommonly is encountered as a result of pDNA vaccination. These includean observed induction of a specific immune response in the majority ofthe immunised animals, while the approaches of using cytokine encodingvectors was considerably less efficient in that regard (data not shown).The enhancement was also associated with a significant quantitativeincrease in the response as compared to giving the pDNA vaccinationalone or in combination with the pDNA expressing cytokine genes, and wasobserved both as increased specific antibody titres and as enhancedproliferative and cytotoxic T-cell responses.

The induction of an adequate immune response requires the participationof multiple components of the immune system, and pDNA immunisationfulfils this requirement as it induces both humoral and cellularresponses including CTL responses, all of which were found to beenhanced by tucaresol. Moreover, while other modes of enhancing pDNAimmunisation will lead to an antibody of a T-cell biased immuneresponse, co-injection of tucaresol resulted in a general enhancement ofboth types of specific immunity including the enhancement of Th1 and Th2associated antibody responses. The combination of pDNA vaccination andtucaresol can therefore be considered in conditions where either acellular of antibody based immune response would be beneficial for thehost.

There was a marked increase in the production of specific antibodyproduction as also measured by the highest IgG:IgM ratio as compared tothe other immunisation procedure (data not shown). This points out thepotential advantage of using this procedure during the production ofmonoclonal antibodies.

The marked ability of tucaresol to enhance pDNA induced specific T-cellresponses to hsp65, Influenza NP and EBNA-4, as detected byproliferation, cytokine production and cytotoxicity, is of particularimportance. Protective immunity to mycobacterial infection is dependenton both CD4⁺ T-cells with the capacity to secrete macrophage activatingcytokines, including IFNγ and on cytotoxic CD8⁺ cells which caneliminate infected macrophages. Protective immunity to EBV infection isdependent on CD4⁺ and CD8⁺ T-cell responses, and T-cell basedimmunotherapy against post-transplant transplant lymphoproliferativedisorders has already proven to be efficient. Since pDNA vaccinationcombined with tucaresol favors both CD4 and CD8 mediated responses, asshown here, this is an attractive mode of vaccination to be applied innew T-cell vaccines against intra-cellular bacteria and viruses.

Tucaresol is a chemically well-defined molecule which has already beenclinically tested. This should simplify the approval procedure of thisdrug in new pDNA-based vaccination protocols. Furthermore, as it wasshown to be systematically active there is no need for localco-administration of pDNA and tucaresol, as shown here by combiningintramuscular pDNA immunisation with subcutaneous injection oftucaresol. The combination of intradermal “ballistic” delivery of pDNAvaccination with oral administration of tucaresol may prove a veryattractive mode of immunisation particularly under conditions whereparenteral immunisations should be avoided due to risks of blood-borneinfections or cultural stigmata associated with injections.

In summary, we present herein data that show for the first time theutility of using a Schiff base forming drug as a simple and effectivemethod to augment the specific immune response induced by pDNAvaccination. These data are applicable for both clinical and industrialsettings.

Experimental Methods

Plasmid Construction and Test

All genes were inserted in the pCDNA3 vector (Invitrogen BV, NV Leek,The Netherlands). Mycobacterium bovis hsp65 cDNA was excised fromplasmid pRIB1300 (kindly provided by Dr. R. v d Zee, Utrecht University,Utrecht, The Netherlands) using Eco RI and Sal I and sub-cloned in theEco RI and Xho I sites of pCDNA3 MCS. The identity and orientation ofthe gene in the resulting plasmid (p3M.65) were confirmed withrestriction mapping. Expression and production of hsp protein wasdetected in the lysate of COS-7 transfected with p3M.65 by lipofectionusing Lipofectine (Life technologies, Paisley, Scotland). Lysates wereelectrophoresed on 12% SDS-PAGE gel, followed by Western Blotting onPVDF membrane (BioRad, CA) and immuno-detection by anti-Mycobacterialhsp65 specific monoclonal antibody DC-16 (kindly provided by Dr. JurajIvanyi, London, U.K.). This was then detected with a secondary alkalinephosphatase conjugated goat anti-mouse Ig (Southern Biotech, AL) and theblot was developed using the western blue substrate system (Promega,Madison, Wis.).

ELISA Procedures

Sera from immunised mice were collected and used in direct ELISA asdescribed earlier. Recombinant mycobacterial hsp65 (kindly provided byDr R. v d Zee) was used at a concentration of 4 μg/ml carbonate bufferto coat wells of 96 well plate (Maxisorp, Nunc, Denmark) overnight at 4°C. Sera were added in duplicate at 1:100 dilution, incubated overnightat 4° C. Binding antibodies were detected using IgG (preabsorbed againstmouse IgM), IgG1 and IgG2a specific alkaline phosphatase conjugated goatanti-mouse sera (Southern Biotech).

Mice

HLA-A2*0201/Kb transgenic mice (kindly provided by Dr L. Sherman,Scripps Laboratories, San Diego, Calif.) used in this study have beendescribed. These mice express a chimeric MHC class II molecule I whichthe α1 and α2 domains are of the HLA-A*0201 molecule while the α3trans-membrane and cytoplasmic domains are of the mouse H2 Kb molecule.This construction permits the binding site of the mouse CD8 molecule onthe T-cell to interact with the α3 domain of the chimeric molecule. Thesurface expression of the HLA-A*0201/Kb was confirmed using HLA-A*0201specific FITC-conjugated monoclonal antibody (One Lambda, Ca) andassessed by flow cytometry using FACSCAN (Becton Dickinson & Co.,Mountain View, Calif.). C575BI/6 mice have been described. These micehave a defined influenza virus nucleoprotein CTL epitope. ACA (H-2f)mice were purchased from Jackson (Jackson Laboratory, Bar Harbor, Main).Mice were propagated and held in our SP environment in MTC animal houseat the Karolinska Institute.

Immunisation

Genetic immunisation was accomplished by intra-muscular immunisation.Plasmids were prepared form LB ampicillin E. coli culture using Qiagenplasmid giga kit (Qiagen GmbH, Hilden, Germany). Concentrations andpurity were determined using spectrophotometer and analytical gelelectrophoresis. Mice were injected in the regeneratingtibialis-anterior muscle according to the method of Davis et al using 20μg pDNA/100 ul/muscle of either control plasmid (P3), EBNA-4 expressingplasmid plus control plasmid p3 (E4), p3M.65 expressing plasmid pluscontrol plasmid p3 (p3M.65), p3M.65 plus GM-CSF expression plasmids(p3M.65G), or p3M.65 plus IFNγ expression plasmid (9p3M.65γ). Plasmidswere mixed in equal molar quantities. Alternatively, immunisation wascarried out by gene gun according to the method of Fynan et al (27)using 10 ng or 100 ng of DNA plasmid encoding A/PR8/34 influenza virusnucleoprotein (pVAC1.PR) or empty vector, or 2 ug of plasmid encodingEBNA-4 (E4) or control vector p3. Tucaresol treated mice were immunisedwith E4, p3M.65, or pVAC1.PR (E4,T p3M.65,t and pVAC1.NP PR(Tuc sc)respectively) and either injected at the same time with 1 mg tucaresoleach sub-cutaneously or received daily injections of tucaresol 200 μgper mouse for a period of 4 days also sub-cutaneously. Mice receivedboosting two weeks after priming with the same dose.

Cell Lines

Jurkat A*0201/Kb(Jk-A2/kb), a human t-cell leukaemia 0HLA-A*0201negative cell line stably transfected with HLA-A80201/Kb chimeric gene(kindly provided by Dr. W. M. Kast, Loyola University, Maywood Ill.).The S6C cell line was derived from a spontaneous mammary adeno-carcinomathat has been originated in an ACA mouse. S6C-gpt and S6C-E4 are controlplasmid and EBNA-4 transfectant respectively (kindly provided by Dr.George Klein, MTC, Karolinska Institute, Stockholm). All cell lines weremaintained by passing in vivo in syngeneic ACA mice and in vitro in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mlpenicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, and 2 mML-glutamine.

Proliferation Test

Splenocytes were harvested from immunised mice. A single cell suspensionwas prepared and cells were re-suspended in IMDM supplemented with 10%FBS and L-glu and antibiotics. Mixed splenocytes and tumor cell cultures(MSTC) were prepared by mixing 3×10⁶ tumor cells per ml. Cultures wereincubated for 5 days at 37° C. in 7.5% CO₂. One μCi of tritium labelledthymidine was added to each well of U-shaped bottom 96 well plates.Cells were further incubated for 18 hours in the same conditions asabove and harvested and the amount of incorporated tritium labelledthymidine was measured using Beta Plate reader (Wallac, Turku, Finland).The test was done in triplicates and the stimulation index (SI) wascalculated using the formula: SI=splenocyte proliferation towards theS6C-EBNA-4 transfectant (or EBNA-4 Vaccinia infected)/splenocyteproliferation toward S6C-gpt (or TK- Vaccinia infected) controltransfectant.

Cytokine assays

Mixed splenocytes (from E4 immunised mice) and tumor cell cultures(MSTC) were prepared by mixing 3×10⁶ splenocytes plus 3×105 tumor cellsper ml. Supernatants were collected after 72 hours of culture and weretested for the presence of interferon gamma (IFNγ) and IL-4 usingcommercially available matched antibody pairs for mouse cytokines ELISA(Immunokontact, Bioggio, Switzerland) according to the manufacturer'sinstructions.

Specific CTL Line Generation and Cytotoxicity Assays.

Peptide specific CTL lines were prepared in 12-well plates as follows.Splenocytes, from immunised or control non-immunised mice were plated at6×10⁶ per well and co-cultured with 3×10⁶ peptide pulsed (5 μg per mlP4) syngeneic splenocytes. After 6–8 days cell-mediated cytotoxicity wasmeasured by ⁵¹Cr release as follows. One million target cells wereincubated at 37° C. in the presence of 200 μCi sodium ⁵¹Cr chromate(Amersham, UK) for 1 hour, washed three times and re-suspended incompete medium at 10⁵ cells/ml in the presence of absence of 10 μg ofthe relevant (P4) or irrelevant (influenza NP 58–66) peptide. The testwas performed by incubating 5×10³ target cells at different effector totarget ratios in triplicate wells at a final volume of 200 μl inV-bottomed 96 well plates. Cells were incubated for 4 hours at 37° C.after which supernatants were harvested and used to determine specificlysis using the following equation: percent specificrelease=100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release).

It is to be understood that the present invention has been described byway of example only, and that modifications and/or alterations thereto,which would be obvious to a skilled person based upon the disclosureherein are also considered to fall within the scope and spirit of theinvention, as defined in the appended claims.

REFERENCES

-   1. Donnelly J. et al, “DNA Vaccines” Annu. Rev. Immunol. 1997, 15:    617–48.-   2. Donnelly, J. J., Friedman A., Martinez D., Montogomery, D. L.,    Shiver, J. W., Motzel, S. L., Ulmer, J. B., Liu, M. A. 1995.    Preclinical efficacy of a prototype DNA vaccine-enhanced protection    against antigenic drift in influenza-virus. Nature Med. 1:583–87.-   3. Lu, S., Arthos, J., Montefiori, D.C., Yasutomi, Y., Manson, K.,    Mustafa, F., Johnson, E., Santoro, J. C., Wissink, J., Mullins, J.    I., Haynes, J. R., Letvin, N. L., Wyand, M., Robinson, H. L. 1996.    Simian immunodeficiency virus DNA vaccine trial in macaques. J.    Virol. 70:3978–91.-   4. Fuller, D. H. Haynes, J. R., 1994. A qualitative progression in    HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral    immune responses in mice receiving a DNA-based glycoprotein 120    vaccine. AIDS Res. Hum. Retrovir. 10:1433–41.-   5. Krieg, A. M., Yi, A-K., Matson, S., Waldschmidt, T. J.,    Bishop, G. A., Teasdale, R., Koretsky, G. A. Klinman, D. M., 1995.    CpG motifs in bacterial DNA trigger direct B-cell activation. Nature    374:546–48.-   6. Messina, J. P., Gilkeson, G. S., Pisetsky, D. S., 1991.    Stimulation of in vitro murine lymphocyte proliferation by bacterial    DNA. J. Immunol. 147:1759–64.-   7. Yamamoto, S., Yamamoto, T., Kataoka, T., Kuramoto, E., Yano, O.,    Tokunaga, T., 1992. Unique palindromic sequences in synthetic    oligonucleotides are required to induce TNF and augment TNF-mediated    natural killer activity. J. Immunol. 148:4072–76.-   8. Tang, D. C., Devit, M., Johnston, S. A., 1992. Genetic    immunisation is a simple method for eliciting an immune response.    Nature 356:152–54.-   9. Yankauckas, M. A., Morrow, J. E., Parker S. E., Abai, A.,    Rhodes, G. H., Dwarki, V. J., Gromkowski, S. H. 1993. Long-term    antinucleoprotein cellular and humoral immunity is induced by    intramuscular injection of plasmid DNA containing NP gene DNA Cell    Biol. 12:771–76.-   10. Wang, B., Boyer, J., Srikantan, V., Coney, L., Carrano, R.,    Phan, C., Merva, M., Dang, K., Agadjanyan, M., Gilbert L., Ugen, K.    E., Williamson, W. V., Weiner, D. B. 1993. DNA inoculation induces    neutralising immune-responses against human-immunodeficiency-virus    type-1 in mice and nonhuman-primates. DNA Cell Biol. 12:799–805.-   11. Cox, G., Zamb, T. J., Babiuk, L. A. 1993. Bovine    herpesvirus-1-immune-responses in mice and cattle injected with    plasmid DNA. J. Virol. 67:5664–67.-   12. Davis, H. L., Michel, M. L., Whalen, R. G., 1993. DNA-based    immunisation induces continuous secretion of hepatitis-b    surface-antigen and high-levels of circulating antibody. Human Mol.    Genet. 2:1847–51.-   13. Ulmer, J. B., Donnelly, J. J. Parker, S. E., Rhodes, G. H,    Felgner, P. L., Dwarki, V. J., Gromokowski, S. H., Deck, R. R.,    Dewitt, C. M., Friedman, A., Hawe, L. A., Leander, K. R., Martinez,    D., Perry, H. C., Shiver, J. W., Montgomery, D. L., Liu, M.    A., 1993. Heterologous protection against influenza by injection of    DNA encoding a viral protein. Science 259:1745–49.-   14. Kuhober, A., Pudollek, H—P, Reifenberg, K., Chisari, F. V.,    Schlicht, H-J, Reimann, J., Schirmbeck, R., 1996. DNA immunisation    induces antibody and cytotoxic T cell-responses to hepatitis B core    antigen in H-2b mice. J. Immunol. 156:3687–95.-   15. Okuda, K., Bukawa, H., Hamajima, K., Kawamoto, S., Sekigawa, K.    I., Yamada, Y., Tanaka, S. I., Ishii, N, Aoki, I., Nakamura, M.,    Yamamoto, H., Cullen, B. R., Fukushima, J., 1995. Induction of    potent humoral and cell-mediated immune-responses following    direct-injection of DNA encoding the HIV type-1 env and rev    gene-products. Aids Res. Human Retrovir. 11:933–43.-   16. Liu, M. A., Davies, M. E., Yasutomi, Y., Perry, H C., Letvin, N.    L., Shiver, J. W., 1994. Immune responses to HIV generated by DNA    vaccines. In Retroviruses of Human AIDS and Related Animal Diseases,    ed. M. Girard, B. Dodet, pp. 197–200. Lyon: Fond. Mercel-Merieux.-   17. Shiver, J. W., Perry, H. C., Davies, M. E., Freed, D. C.,    Liu, M. A., 1995. Cytotoxic T-lymphocyte and helper T cell responses    following HIV polynucleotide vaccination. Ann. N.Y. Acad. Sci.    772:198–208.-   18. Xiang, Z. Q., Ertl, H., 1995. Manipulation of the    immune-response to a plasmid-encoded viral-antigen by coinoculation    with plasmids expressing cytokines. Immunity 2:129–35.-   19. Conry, R. M., Widera, G., Lobuglio, A. F., Fuller, J. T.,    Moore, S. E., Barlow, D. L., Turner, J., Curiel, D. T., 1996.    Selected strategies to augment polynucleotide immunisation. Gene    Therapy 3:67–74.-   20. Tascon, R., Stavropoulos, E., Colston, M. J.,    Lowrie, D. B. 1996. Polynucleotide vaccination induces a significant    protective immune response against Mycobacteria. In Vaccines 96,    ed. F. Brown, R. M. Chanock, M. S. Ginsbert, R. A. Lerner, pp.    45–49. Cold Spring Harbor, N.Y.: Cold Spring Harbor Lab. Press.-   21. Maniatis, T., Sambrook, J., Fritsch, E. F., “Molecular cloning:    A Laboratory Manual”, Cold Spring Harbour Laboratory, Cold Spring    Harbour Press, Vols 1–3, 2^(nd) Edition, 1989.-   22. Rhodes et al, “Therapeutic Potentiation of the Immune System by    Co-Stimulatory Schiff-Based-Forming Drug,” Nature, 377, pp 71–75,    1995.-   23. Brohm W et al, “Routes of Plasmid DNA Vaccination that Prime    Murine Humoral and Cellular Immune Reponses,” Vaccine, Vol 16, No.    9/10, pp 949–954, 1998.-   24. Remington's Pharmaceutical Sciences, Mack Publishing Company,    Easton, Pa. (1985).-   25. Vordermeier, H. M., Coombs, A. G. A., Jenkins, P. McGee, J. P.,    O'Haga, D. T. Davis, S. S. and Singh, M. Synthetic delivery systems    for tuberculosis vaccines: immunolical evaluation of the M.    tuberculosis 38 kDa protein entrapped in biodegradable PLG    microparticles. Vaccine 13: 1576–1582 1995.-   26. Haynes J R. McCabe DE. Swain W F. Wedera G. Fuller J T.    Particle-mediated nucleic acid immunisation. Journal of    Biotechnology. 44: 37–42, 1996.-   27. Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes J. T.,    Santoro, J. C., and Robinson, H. L., (1993) DNA vaccines: protective    immunisations by parenteral, mucosal and gene-gun inoculatiions.    Proc. Natl. Acad. Sci. USA 90:11478

1. A method of vaccinating a mammal against a disease state, comprisingadministrating to said mammal, within an appropriate vector, anucleotide sequence encoding an antigenic peptide associated with thedisease state and not associated with a virus particle; additionallyadministering to said mammal a Schiff base forming compound whichenhances both humoral and cellular immune responses initiated by theantigenic peptide, the compound being selected from the group consistingof: 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-(2-formyl-3-hydroxyphenoxy)pentanamide; N,N-diethyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; N-isopropyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; ethyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;(±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid; methyl3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid; benzyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide; ethyl4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(3-acetylamino-2-fomyl phenoxy)pentanoic acid; Aminoguanidine;4-(2-formyl-3-hydroxyphenoxy)butanoic acid;6-(2-formyl-3-hydroxyphenoxy)hexanoic acid; ethyl4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;5-(2-formyl-3-hydroxy-4-methoxyphenoxy)pentanoic acid;3-(2-formyl-3-hydroxyphenoxy)propionitrile; 4-Hydroxyphenylacetaldehyde;Phenylacetaldehyde; 4-Methoxyphenylacetaldehyde;1-hydroxy-2-phenylpropane; 3-Phenylproponionaldeyde;4-Nitrobenzaldehyde; Methyl 4-formylbenzoate; 4-Chlorobenzaldehyde;4-Methyloxybenzaldehyde; 4-Methylbenzaldehyde; 8,10-Dioxoundecanoicacid; 4,6-Dioxoheptanoic acid; Pentanedione; 5-methoxy-1-tetralone;6-methoxy-1-tetralone; 7-methoxy-1-tetralone; 2-tetralone;3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one; Naringenin4′,5,6-trihydroxyflavonone; 4′-methoxy-2-(4-methoxyphenyl)acetophenone;6,7-dihydroxycoumarin; 7-methoxy-2-tetralone; 6,7-dimethoxy-2-tetralone;6-hydroxy-4-methylcoumarin; Homogentisic acid gamma lactone;6-hydroxy-1,2-naphthoquinone; 8-methoxy-2-tetralone; and physiologicallyacceptable salts thereof, where appropriate.
 2. The method according toclaim 1 wherein administration of the compound takes place on betweenone and seven occasions, between 14 days prior to and 14 days postadministration of the nucleotide sequence.
 3. The method according toclaim 1 wherein administration of the compound takes place on betweenone and seven occasions, between 7 days prior to and 7 days postadministration of the nucleotide sequence.
 4. The method according toclaim 1 wherein administration of the compound takes place between 24hours prior to and 24 hours post administration of the nucleotidesequence.
 5. The method according to claim 1 wherein administration ofthe compound is simultaneous with administration of the nucleotidesequence.
 6. The method according to claim 1 wherein administration ofthe compound and the nucleotide sequence is repeated between 1 and 4times, at intervals of between 1 day and about 18 months.
 7. The methodaccording to claim 1 wherein administration of the nucleotide sequenceis via the oral, nasal, pulmonary, intramuscular, subcutaneous orintradermal route.
 8. The method according to claim 7 wherein thenucleotide sequence is administered using a gene-gun delivery technique.9. The method according to claim 1 wherein administration of thecompound is via the oral, nasal, pulmonary, intramuscular, subcutaneous,intradermal or topical route.
 10. The method according to claim 9wherein the compound is administered using a gene-gun deliverytechnique.
 11. The method according to claim 1 wherein the compound isadministered at a dose of between 0.1 mg/kg and 100 mg/kg peradministration.
 12. The method according to claim 1 wherein the mammalis a human.
 13. The method according to claim 1 wherein the compound is4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid.
 14. A combination ofcomponents for separate, sequential or concomitant administration in amethod of vaccinating a mammal against a disease state, comprisingadministrating to said mammal, within an appropriate vector, anucleotide sequence encoding an antigenic peptide associated with thedisease state and not associated with a virus particle; additionallyadministering to said mammal a Schiff base forming compound whichenhances both humoral and cellular immune responses initiated by theantigenic peptide, the compound being selected from the group consistingof: 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-(2-formyl-3-hydroxyphenoxy)pentanamide; N,N-diethyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; N-isopropyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; ethyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;(±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid; methyl3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid; benzyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide; ethyl4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(3-acetylamino-2-fomyl phenoxy)pentanoic acid; Aminoguanidine;4-(2-formyl-3-hydroxyphenoxy) butanoic acid;6-(2-formyl-3-hydroxyphenoxy)hexanoic acid; ethyl4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;5-(2-formyl-3-hydroxy-4-methoxyphenoxy) pentanoic acid;3-(2-formyl-3-hydroxyphenoxy)propionitrile; 4-Hydroxyphenylacetaldehyde;Phenylacetaldehyde; 4-Methoxyphenylacetaldehyde; jkhu1-hydroxy-2-phenylpropane; 3-Phenylproponionaldeyde;4-Nitrobenzaldehyde; Methyl 4-formylbenzoate; 4-Chlorobenzaldehyde;4-Methyloxybenzaldehyde; 4-Methylbenzaldehyde; 8,10-Dioxoundecanoicacid; 4,6-Dioxoheptanoic acid; Pentanedione; 5-methoxy-1-tetralone;6-methoxy-1-tetralone; 7-methoxy-1-tetralone; 2-tetralone;3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one; Naringenin4′,5,6-trihydroxyflavonone; 4′-methoxy-2-(4-methoxyphenyl)acetophenone;6,7-dihydroxycoumarin; 7-methoxy-2-tetralone; 6,7-dimethoxy-2-tetralone;6-hydroxy-4-methylcoumarin; Homogentisic acid gamma lactone;6-hydroxy-1,2-naphthoquinone; 8-methoxy-2-tetralone; and physiologicallyacceptable salts thereof, where appropriate; wherein the combinationcomprises the nucleotide sequence encoding for an antigenic peptide andthe compound which enhances both humoral and cellular immune responsesinitiated by the antigenic peptide.
 15. A method of vaccinating a mammalagainst a disease state, comprising administrating to said mammal,within an appropriate vector, a nucleotide sequence encoding anantigenic peptide associated with the disease state; additionallyadministering to said mammal a Schiff base forming compound whichenhances at least Th1 and Th2 associated responses initiated by theantigenic peptide, the compound being selected from the group consistingof: 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-(2-formyl-3-hydroxyphenoxy)pentanamide; N,N-diethyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; N-isopropyl5-(2-formyl-3-hydroxyphenoxy)pentanamide; ethyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-(2-formyl-3-hydroxyphenoxy)pentanonitrile;(±)-5-(2-formyl-3-hydroxyphenoxy)-2-methylpentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-2,2-dimethylpentanoic acid; methyl3-(2-formyl-3-hydroxyphenoxy)methylbenzoate;3-(2-formyl-3-hydroxyphenoxy)methylbenzoic acid; benzyl5-(2-formyl-3-hydroxyphenoxy)pentanoate;5-[4-(2-formyl-3-hydroxyphenoxy)-N-butyl]tetrazole;7-(2-formyl-3-hydroxyphenoxy)heptanoic acid;5-(2-formyl-3-hydroxy-4-n-propoxyphenoxy)pentanoic acid;5-(4,6-dichloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(2-formyl-3-hydroxyphenoxy)-N-methylsulphonylpentanamide; ethyl4-(2-formyl-3-hydroxyphenoxymethyl)benzoate;5-(4-chloro-2-formyl-3-hydroxyphenoxy)pentanoic acid;5-(3-acetylamino-2-fomyl phenoxy)pentanoic acid; Aminoguanidine;4-(2-formyl-3-hydroxyphenoxy)butanoic acid;6-(2-formyl-3-hydroxyphenoxy)hexanoic acid; ethyl4-(3-acetylaminio-2-formylphenoxymethyl)benzoate;4-(3-acetylamino-2-formylphenoxymethyl)benzoic acid;2-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid;5-[4-(2-formyl-3-hydroxyphenoxymethyl)phenyl]tetrazole;5-(2-formyl-3-hydroxy-4-methoxyphenoxy)pentanoic acid;3-(2-formyl-3-hydroxyphenoxy)propionitrile; 4-Hydroxyphenylacetaldehyde;Phenylacetaldehyde; 4-Methoxyphenylacetaldehyde;1-hydroxy-2-phenylpropane; 3-Phenylproponionaldeyde;4-Nitrobenzaldehyde; Methyl 4-formylbenzoate; 4-Chlorobenzaldehyde;4-Methyloxybenzaldehyde; 4-Methylbenzaldehyde; 8,10-Dioxoundecanoicacid; 4,6-Dioxoheptanoic acid; Pentanedione; 5-methoxy-1-tetralone;6-methoxy-1-tetralone; 7-methoxy-1-tetralone; 2-tetralone;3-hydroxy-1-(4-methoxyphenyl)-3-methyl-2-butanone;2′,4′-dihydroxy-2-(4-methoxyphenyl)acetophenone;2-hydroxy-1-(4-methyoxyphenyl)-pent-2ene-4one; Naringenin4′,5,6-trihydroxyflavonone; 4′-methoxy-2-(4-methoxyphenyl)acetophenone;6,7-dihydroxycoumarin; 7-methoxy-2-tetralone; 6,7-dimethoxy-2-tetralone;6-hydroxy-4-methylcoumarin; Homogentisic acid gamma lactone;6-hydroxy-1,2-naphthoquinone; 8-methoxy-2-tetralone; and physiologicallyacceptable salts thereof, where appropriate.
 16. The method according toclaim 15 wherein the compound is4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid.
 17. The methodaccording to claim 1 wherein the vector which comprises the nucleotidesequence encoding the antigenic peptide is administered in a naked form.18. The method according to claim 1 wherein the vector which comprisesthe nucleotide sequence encoding the antigenic peptide is encapsulatedby liposomes or within polylactide co-glycolide particles.
 19. Thecombination according to claim 14 wherein the vector which comprises thenucleotide sequence encoding the antigenic peptide is administered in anaked form.
 20. The combination according to claim 14 wherein the vectorwhich comprises the nucleotide sequence encoding the antigenic peptideis encapsulated by liposomes or within polylactide co-glycolideparticles.
 21. The method according to claim 15 wherein administrationof the compound takes place on between one and seven occasions, between14 days prior to and 14 days post administration of the nucleotidesequence.
 22. The method according to claim 15 wherein administration ofthe compound takes place on between one and seven occasions, between 7days prior to and 7 days post administration of the nucleotide sequence.23. The method according to claim 15 wherein administration of thecompound takes place between 24 hours prior to and 24 hours postadministration of the nucleotide sequence.
 24. The method according toclaim 15 wherein administration of the compound is simultaneous withadministration of the nucleotide sequence.
 25. The method according toclaim 15 wherein administration of the compound and the nucleotidesequence is repeated between 1 and 4 times, at intervals of between 1day and about 18 months.
 26. The method according to claim 15 whereinadministration of the nucleotide sequence is via the oral, nasal,pulmonary, intramuscular, subcutaneous or intradermal route.
 27. Themethod according to claim 26 wherein the nucleotide sequence isadministered using a gene-gun delivery technique.
 28. The methodaccording to claim 15 wherein administration of the compound is via theoral, nasal, pulmonary, intramuscular, subcutaneous, intradermal ortopical route.
 29. The method according to claim 28 wherein the compoundis administered using a gene-gun delivery technique.
 30. The methodaccording to claim 15 wherein the compound is administered at a dose ofbetween 0.1 mg/kg and 100 mg/kg per administration.
 31. The methodaccording to claim 15 wherein the mammal is a human.
 32. The methodaccording to claim 15 wherein the vector which comprises the nucleotidesequence encoding the antigenic peptide is administered in a naked form.33. The method according to claim 15 wherein the vector which comprisesthe nucleotide sequence encoding the antigenic peptide is encapsulatedby liposomes or within polylactide co-glycolide particles.